Original article

Evaluation of heavy metal contamination and accumulation in lettuce (Lactuca sativa L.) plants

Ambar Rosa Guzmán-Morales^{1  *} 
http://orcid.org/0000-0003-3834-3546

Orestes Cruz-La Paz¹ 
http://orcid.org/0000-0002-6129-4976

Ramiro Valdés-Carmenate¹ 
http://orcid.org/0000-0002-4244-1950

Pedro A. Valdés-Hernández¹ 
http://orcid.org/0000-0002-8570-0895

¹Universidad Agraria de La Habana “Fructuoso Rodríguez Pérez”, carretera a Tapaste y Autopista Nacional, San José de las Lajas, Mayabeque, Cuba, CP 32 700

ABSTRACT

Lettuce is a plant species considered to be a bioindicator of the presence of heavy metals in soils, consumed fresh by the population, with the highest concentrations found in its leaves. The research was carried out in areas surrounding the White Ceramic Enterprise "Adalberto Vidal" of the Popular Council of Jamaica, in San José de las Lajas, Mayabeque, Cuba, with the aim of evaluating the extraction of heavy metals by lettuce plants under production conditions. A randomized sampling of soil and crop was carried out. The content of heavy metals was determined by X-Ray Fluorescence-Energy Dispersive X-Ray and the transfer coefficient in the crop was estimated. Values obtained were compared with the maximum permissible limits and hazardous levels for soil and plants, proposed in the literature. Results showed that the soil is contaminated by Ni, Cu and Pb metals. The accumulation of seven heavy metals studied (Fe, Co, Zn, Mn, Cu, Ni and Pb) was found in the crop at high levels and the highest accumulation is in the aerial part, which represents a danger for local food safety.

Key words: degradation; pollution; toxicity

INTRODUCTION

One plant species considered to be a bioindicator of heavy metals is lettuce (Lactuca sativa L.), which is used fresh by the population and it is in the edible part (leaves) where the highest concentration of these elements accumulates ¹. Despite the effort, in terms of environmental education, that has been made by the FITOPLANT Scientific Group of the UNAH (Agrarian University of Havana), even in the areas surrounding the White Ceramic Enterprise "Adalberto Vidal" (current joint venture since 2018 SANVIG, S.A.) of the Popular Council of Jamaica, this crop continues to be harvested for food and marketing purposes, since it does not show symptoms of toxicity under conditions of metallic stress ².

Some plants and other organisms develop a complex physiological mechanism to minimize the negative effects of heavy metals, controlling the absorption, accumulation and translocation of these elements in the plant tissue. These mechanisms protect cells, causing plant tolerance to these elements, so that treatment techniques have their genesis in the ability of different organisms (plants and microorganisms) to degrade, extract or immobilize organic or inorganic contaminants in soil or water ³. Therefore, studies attributed to the presence of heavy metals and some of their compounds in agricultural areas located over old smelters, refineries, aluminum factories, metallurgical industry and ceramic companies are important. Some of them precipitate in the sewage sludge, so the crop usually contains high concentrations of these metals ⁴.

It was scientifically demonstrated that in addition to causing some of the most serious environmental problems, exposure to heavy metals in certain circumstances is the cause of direct damage to humans, although they are considered trace elements, they are food toxins and their negative effects on health are manifested in the long term.

The action of different heavy metals has been reported on the increase of cancer cases, nervous system lesions and intellectual and mental retardation as a result of the consumption of water and food contaminated with these metals ^5-8.

The aim of this study is to evaluate the heavy metal extraction capacity of lettuce plants under production conditions in the areas surrounding the wastewater discharge of the "Adalberto Vidal" Ceramic Enterprise in San José de las Lajas municipality.

MATERIALS AND METHODS

The study was carried out in an agricultural area adjacent to the discharge of waste from the White Ceramic Enterprise "Adalberto Vidal" in San José de las Lajas municipality, Mayabeque province (Figure 1), located at a distance of 200 m from the source of contamination and a profile of the "La Asunción" Farm, located in the same municipality, in an area not affected by waste, was established as the standard soil, it is a land not cultivated for more than 60 years, with spontaneous vegetation and does not receive any treatment.

Figure 1 a) Expulsion and transit of the Enterprise's residuals b) Production area 

A completely randomized design was used to set up the experiment. For the matrices under study (soil and crop) five sampling points were taken with three replicates. Sampling was carried out following diagonals in the plot: beginning, middle and end, obtaining at the end a single sample as an average of sampling points.

Soil samples were taken manually at 0-20 cm depth, disregarding the surface layer, with an approximate separation of 1 m from the plot boundary. Samples of the lettuce crop (Lactuca sativa L.) var. Black Simpson were taken in this same condition at the time of harvest, carrying out determinations of heavy metals in root, stem and leaves. In addition, the transfer coefficient was calculated from the aerial part/root part ratio, which indicates where the metal ions are translocated to.

Soil samples were cleaned (removal of stones, plants, foreign matter) and sieved at 125 μm and then mixed with cellulose in a 4:1 ratio, after homogenization, "infinitely coarse" tablets were made up, pressed at 15 t. The irradiation of the pellets was carried out for 6 h of live time and the dead time was in all cases below 1 %.

The analyses were performed at the Analytical Laboratory of the Nuclear Physics Department of InSTEC by Energy-Dispersive X-Ray Fluorescence ⁹. The determination of heavy metal concentration was performed with a high-resolution Si(Li) detector for X-ray spectrometry and a ²³⁸Pu radioisotope source of low-energy photons by Energy-Dispersive X-Ray Fluorescence. An aluminum excitation chamber with a collimator for the characteristic radiation of 1 cm diameter and adjustable sample holder height was used. The minimum source-detector distance is 18 mm, direct radioisotope excitation was provided by AMERSHAND, which has a half-life of 87 years, an emission energy (U-L) of 12-17 KeV, with an α decay scheme. All spectra were processed with WinAxil code version 4.5.2.

For the determinations in vegetable samples of lettuce crop, after drying at 70 ºC, the aerial and root parts were separately homogenized by grinding them. Subsequently, 0.1 g of each part was weighed on an analytical balance (Sartorius BP121S) and subjected to a digestion process with HNO3, HCl (3:1) for 30 min. Levels of metals were determined by Atomic Absorption Spectrophotometry with air-acetylene flame in BuckScientific 210 VGP equipment, according to the procedure established and validated in the Environmental Analysis Laboratory ¹⁰.

The STATGRAPHICS Plus statistical program for Windows 5.1 was used for the analysis of the experimental data. The simple ANOVA test was performed, for which a significance level of 0.05 was established for a 95 % confidence interval. The Duncan's comparison were applied to determine between which levels the significant difference was established and for those mean values that presented significant differences, different letters were assigned to them.

In the case of the study of the soil samples, the statistical processing of the data consisted of calculating the confidence intervals of the means by treatments of the variables evaluated, for a confidence level of 95 %.

RESULTS AND DISCUSSION

Concentration of heavy metals in soil

Soil analysis results of samples in the agricultural areas surrounding the discharge of waste from the former White Ceramic Enterprise "Adalberto Vidal" in San José de las Lajas municipality showed that concentrations of the heavy metals evaluated (Cr, Co, Ni, Cu, Zn, Pb and Fe) were higher than those determined in the standard soil (Table 1); with the exception of Co, where the difference is five units, and Fe, 1.41 percentage units, since for this element we refer to the percentage of its quantity in the sample, because it was selected as the reference metal for the study; little influenced by anthropogenic sources, due to the high natural levels of this element in the earth's crust

^11-13.

Table 1 Concentration of heavy metals (mg kg^-1) in soil that receives the waste from the White Ceramic Enterprise "Adalberto Vidal" in San José de las Lajas municipality  

Samples	Cr ± std	Co ± std	Ni ± std	Cu ± std	Zn ± std	Pb ± std	Fe(%)± std
	(mg kg^-1)
Pattern	118±42	15±4	84±29	364±98	117±40	90±27	4,54±1
Polluted	154±38	20±38	317±25	421±32	415±24	173±31	5,95±1
RV^a	100	9	35	36	140	85
IV^a	380	240	210	190	720	530
UPL^b	100	50	75	100	300	100
EC^c	100	25	74	55	70	13	5

a- Reference Values (RV) and Intervention Values of the Dutch Norms ¹¹

b- Upper Permissible Limit in soils ¹²

c- Reported values for the Earth's Crust ¹²

The concentration values of heavy metals in the contaminated soil are higher than the reference values, and in the case of nickel and copper, the value exceeds the intervention limit of these standards, which classifies the soil as moderately contaminated in Cr, Co, Zn and Pb and in need of urgent remediation, due to Ni and Cu concentrations. In turn, these values, with the exception of cobalt, are higher than those reported for the earth's crust and those proposed as Upper Permissible Limits ¹².

For the rest of the elements analyzed, the decreasing order of contamination contributions is Zn, Ni, Pb, Cu and C, with values of 298, 233, 83, 57 and 36 units of differences respectively between the contaminated soil and the standard soil, which is equivalent to a percentage of 99.33 % for Zn and 310.66 % for Ni above the upper permissible limit, which are the ones that contribute the most ¹².

These results are directly related to the wastes coming from the White Ceramic Enterprise "Adalberto Vidal", which has among its materials all kinds of clays and kaolins, coming from different provinces of the country: Pinar del Río (Club de Cazadores), Las Tunas (Dumañuelos), Isla de la juventud (Santa Elena), as well as feldspars coming from Sancti Spíritus (Pico tuerto), besides, many other oxides resistant to high temperatures, such as corundum (Al₂O₃), zirconium oxide (ZrO₂) or silicon carbide (SiC) and organic compounds such as methylene blue; clays containing heavy metals in the form of oxides such as: Iron (Fe), Manganese (Mn), Aluminum (Al), Silicon (Si), Barium (Ba), Titanium (Ti), Zirconium (Zr), and Zinc (Zn), among other elements such as Calcium (Ca), Magnesium (Mg) and Sodium (Na) (1,7,10).

The metals with the highest concentrations in the soil according to the calculations are Ni, Cu and Pb (Figure 2); it can be seen that these elements are found in concentrations higher than those required by these established levels, obtaining statistically significant differences with respect to the three values with which they are compared.

Figure 2 Concentration of Ni, Cu and Pb compared to average levels acceptable for healthy food production and phytotoxic levels 

Concentrations of these elements (Ni, Cu and Pb) are higher than those reported for the average of Cuban agricultural soils ^12-16. When comparing the concentration of these metals (Ni, Cu and Pb) with the permissible levels for healthy food (P.L.H.F), phytotoxic average levels (P.A.L) and levels in Cuban agricultural soils (L.A.S Cuba).

All these results have a chemical-biological influence on the crops produced in these areas, since, as it can be verified, the values with which they are compared are related to food safety, a property that takes interest in local food safety ¹⁶. Besides, ratifying the importance of these studies, because the chemical elements that are studied in their majority are essential micronutrients for the growth and development of plants ^13,15. That is why the producer appreciates a growth and development of plants, which does not show symptoms of nutritional deficiency.

Concentration of heavy metals in lettuce crops

When analyzing the concentration values obtained in the lettuce samples, in relation to some permissible levels, it can be seen that the crop is capable of extracting several toxic elements (Fe, Co, Zn, Mn, Cu, Ni, Pb), exceeding in all levels of sufficient or normal concentration in plants (S) ^4,7,12 and also exceeding the excessive or toxic concentrations (E) for all the metals evaluated, except for Mn and Pb (Table 2).

Table 2 Heavy metal concentration (mg kg^-1 of DM) in lettuce crop and its transfer coefficient value 

Samples		Fe	Co	Zn	Mn	Cu	Ni	Pb
		mg kg^-1 of DM
Leaves		974	28	800	114	176	124	174
Roots		1 098	25	1 145	47	321	93	153
Stems		4 720	30	637	243	320	160	133
Permissible limits in plants	S	--	--	30	30	5	0,1-5	--
	T	--	--	50	300	5-20	10	10
	E	--	1	100	1 000	20-100	10-100	30-300
Transfer coefficient		5,19	2,32	1,26	7,6	1,55	2,94	2,01

(S) Sufficient or normal concentration levels in plants, (T) Tolerable levels in agricultural crops, (E) Excessive or toxic concentrations

There is a high accumulation of Fe, Zn and Cu in roots, stems and leaves, with a higher presence of Fe and Zn, which are elements considered toxic for agricultural crops ¹⁷. For the lettuce crop it is not harmful, since it possesses physiological mechanisms that allow it to take them quickly and accumulate them in its aerial parts, making it possible for it to survive in the metallic stress conditions where the plant develops ^18,19. Therefore, it represents an economic reference crop model to study metal accumulation mechanisms, as well as the phytoextraction mechanisms used by phytoremediation, as a method to remediate areas vulnerable to heavy metal contamination ¹⁸.

In the case of the calculated transfer coefficient (Table 2), it was observed that for all the elements evaluated, the translocation is from the radical part to the aerial part, which corroborates the risk of cultivation in contaminated conditions ¹⁹, as it does not comply with food safety ¹⁸. These pollution conditions by heavy metals in soils should not be allowed for the cultivation of agricultural species with food purposes ^20,21, since in the case of lettuce there is a great accumulation of these elements very high in leaves, which is precisely its organ of consumption, besides fresh form and it is classified according to literature, as a bioaccumulator and bioindicator of heavy metals ²².

CONCLUSIONS

• The soils surrounding the old White Ceramic Enterprise's landfill in San José de las Lajas municipality are moderately contaminated with Cr, Co, Zn and Pb and in need of urgent remediation due to Ni and Cu concentrations, which shows that these are areas that are not suitable for agricultural use.

• The values of heavy metals in soil with the highest Ni, Cu and Pb concentration, exceed the permissible levels for healthy food, the average phytotoxic levels and the levels in Cuban agricultural soils.

• The lettuce crop, obtained under conditions of contamination by heavy metals, is shown as an accumulator of Fe, Co, Zn, Mn, Cu, Ni, Pb metals, exceeding in all levels of sufficient or normal concentration in plants.

• The transfer coefficient calculated for the lettuce crop indicates that all the metals studied are translocated from the root to the aerial part of the plant, being higher in order in the case of Mn, Fe, Ni, Co, Pb.

RECOMMENDATIONS

Continuing the physiological-biochemical studies of the species that are cultivated in other areas surrounding the dumping of waste from old White Ceramic Enterprise in San José de la Lajas municipality, to verify their level of toxicity, in order to warn about the danger to food safety in the locality.

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Received: June 24, 2020; Accepted: August 26, 2021

^*Author for correspondence: [e-mail]